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Methods Mol Biol. 2020;2052:351-372. doi: 10.1007/978-1-4939-9748-0_20.

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers.

Author information

1
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA.
2
Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.
3
Department of Pathobiology, University of Illinois College of Veterinary Medicine, Urbana, IL, USA.
4
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA. sibley@wustl.edu.

Abstract

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.

KEYWORDS:

Air-liquid interface; Cryptosporidium parvum; Long-term growth; Primary cell; Transwell

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